Supplementary Table 1 from Identification of Potential Human Oncogenes by Mapping the Common Viral Integration Sites in Avian Nephroblastoma

Supplementary Materials from Industasis, a Promotion of Tumor Formation by Nontumorigenic Stray Cells

(A) Comparison of the extent of apopotosis induced by HHT (after 24hrs exposure) in HHT-resistant (HHT-R) clones compared to the original cell lines (CTRL). (B) Western blot analysis demonstrates upregulation of BCL-XL protein in HHT-R clones compared to CTRL cell lines.

Downregulation of MYC, cFLIP or BCL6 in OCI-Ly7 and BJAB DLBCL cell lines (by western blot) after exposure to HHT.

Western blot analysis showing protein expression profile of key regulators of apoptosis in 18 DLBCL cell lines (12 germinal center B-cell-like (GCB) and 6 activated B-cell-like (ABC) DLBCL cell lines). *UPF4D cell line (GCB origin) was derived in our laboratory.

Cell of origin (COO), BCL2 expression status and IC100 for ABT-199 (µM) and HHT (nM) are shown.

To investigate whether the expression level of BCL2 proteins detected by western blot is comparable with the expression level assessed by IHC analysis, we analyzed protein expression (by IHC) of BCL2, MCL1 and BCL-XL from subcutaneous lymphoma xenografts obtained from mice xenotransplanted with selected DLBCL cell lines with known protein expression (by western blot).

Viability of DLBCL cell lines cultured with different concentrations of HHT (nM) or ABT-199 (µM) was measured by WST-8 survival/proliferation assay for 7-14 days. Graphs indicate % of viable cells, i.e. % of maximal absorbance of untreated cells shown with dashed lines. Representative example of two independent experiments is shown. Standard deviations were < 5% for all measurements.

Shown are examples of different levels of semi-quantitative protein expression (0-3).

The graphs show the extent of apoptosis of DLBCL cell lines induced by different concentrations of HHT, ABT-199 and ABT-737 after 24hrs exposure to particular agents.

Supplemental Figure 1. Next-generation sequencing of PDXs and primary MCL cells Supplemental Figure 2. IHC analysis of primary MCL samples and murine xenografts Supplemental Figure 3. Array comparative genomic hybridization of 24 primary MCL samples Supplemental Figure 4. Sensitivity of HBL2 and MAVER-1 cells resistant to venetoclax to BCL-XL inhibitors WEHI-539 and A1155463 Supplemental Table 1. Complete list of protein coding variants Supplemental Table 2. FISH analyses of the established murine PDXs of MCL Supplemental Table 3. Baseline characteristics of patients Supplemental Table 4. IHC analysis of MCL samples and xenografts Supplemental Table 5. Tumor and spleen weights at the end of in vivo experiments

Gene inactivation of the cyclin‑dependent kinase inhibitors p16INK4a, p15INK4b and p21WAF is frequently mediated by promoter gene methylation, whereas histone deacetylases (HDACs) control gene expression through their ability to deacetylate proteins. The effect of suberohydroxamic acid (SBHA) and 5‑Aza‑2'‑deoxycytidine (Decitabine) (DAC) treatments on the transcription of CDKN2A, CDKN2B and CDKN1A genes, and their effects on molecular biological behavior were examined in two myeloma cell lines, RPMI8226 and U266, which differ in p53‑functionality and IL‑6 expression. In both tested myeloma cell lines, a non‑methylated state of the CDKN2B gene promoter region was detected with normal gene expression, and the same level of p15INK4b protein was detected by immunocytochemical staining. Furthermore, in myeloma cells treated with SBHA and DAC alone, the expression of both p15INK4b and p21WAF was significantly upregulated in RPMI8226 cells (p53‑functional, without IL‑6 expression), whereas in the U266 cell line (p53 deleted, expressing IL‑6) only p21WAF expression was significantly increased. Moreover, the analysis revealed that treatment with DAC induced DNMT3B enhancement in U266 cells. In conclusion, in myeloma cells with IL‑6 expression, significantly increased DNMT3B expression indicated the tumorigenic consequences of 5‑Aza‑2'deoxycytidine treatment, which requires careful use in diseases involving epigenetic dysregulation, such as multiple myeloma (MM).

218 Main document: 4303 (Introduction, Methods, Results, Discussion) Figures: 5 Tables: 1 Supplemental data for review: Supplemental Figures: 4 Supplemental Tables: 5 Translational Relevance: BCL2-targeting agent venetoclax (VTX) has promising anticancer activity in mantle cell lymphoma (MCL), but remissions tend to be short, which calls for rational drug combinations. We demonstrated that MCL1 and NOXA play important roles in mediating resistance to VTX. Consequently, we proposed an experimental treatment strategy based on co-targeting BCL2 with VTX and MCL1 with a highly specific small molecule MCL1 inhibitor S63845,. The combination of VTX and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Research. on May 2, 2021. © 2019 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 19, 2019; DOI: 10.1158/1078-0432.CCR-18-3275

Abstract Purpose: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. Experimental Design: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. Results: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Conclusions: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Mature B-cell malignancies represent the most common types of hematologic tumors. Despite this, little information is available on the composition and function of hematopoiesis in patients with these malignancies. Hematopoietic stem and progenitor cells (HSPCs) might be influenced by at least three key factors: 1. HSPCintrinsic mutations (that might predispose subjects to the development of these malignancies), 2. Direct or indirect impact of malignant lymphocytes present in the bone marrow (BM) or extramedullary tissue, and 3. Age-related changes. The aim of the study herein was to analyze HSPC content in patients with thus far untreated mature B-cell malignancies. We first confirmed the age-related changes observed by Kuranda et al. in our cohort of 22 control samples. While the absolute numbers of hematopoietic stem cells (HSCs) did not change, we observed a negative correlation of their relative numbers with increasing age (Figure 1A,C). The absolute, but not relative numbers of multipotent progenitors (MPPs) positively correlated with age (Figure 1A,C). The most statistically significant change was observed in the compartment of multilymphoid progenitors (MLPs), where both the absolute and relative frequencies positively correlated with age (Figure 1A,C). Both the absolute and relative numbers of pro-B cells were significantly lower in the control samples of the elderly (Figure 1A,C). In addition to the age-related changes, we have recently demonstrated that healthy Caucasians have significantly increased proportions of BM-derived pro-B cells compared to Asians. To avoid any potential ageor race-related biases in HSPC frequencies, the control cohort used in this study comprised BM samples obtained from age-matched healthy Caucasians (all patients were Caucasians as well). The flow cytometry gating strategy is explained in detail in the Online Supplementary Materials and Methods. All patient samples were obtained after written informed consent according to the Helsinki Declaration of 1975 (revised in 1985). The study was approved by the Ethics Committee of the Charles University General Hospital in Prague. The patient cohort (n=125, median age 65 years) included samples of chronic lymphocytic leukemia (CLL, n=21), diffuse large B-cell lymphoma (DLBCL, n=35), follicular lymphoma (FL, n=24), mantle cell lymphoma (MCL, n=27), and multiple myeloma (MM, n=18). Surprisingly, most of the age-related changes observed among control samples were not discernible in patient samples (Figure 1B,D). The exceptions to the rule were absolute and relative frequencies of pro-

Cardiovascular involvement represents a leading cause of mortality and morbidity in sickle cell disease (SCD). Apelin is a peptide involved in the regulation of cardiovascular function.To determine serum apelin among 40 children and adolescents with SCD compared with 40 healthy controls and assess its relation to markers of hemolysis, iron overload as well as cardiopulmonary complications.SCD patients, in steady state and asymptomatic for heart disease, were studied stressing on hydroxyurea/chelation therapy, hematological profile, serum ferritin and apelin levels. Full echocardiographic study including assessment of biventricular systolic function and pulmonary artery pressure was done.Apelin levels were significantly lower in SCD patients compared with controls (P < 0.001). Cardiopulmonary complications were encountered in 30% of patients. Apelin was significantly decreased among patients with cardiopulmonary disease (P = 0.006) whether those at risk of pulmonary hypertension (P = 0.018) or patients with heart disease (P = 0.043). Hydroxyurea-treated patients had higher apelin levels than untreated ones (P = 0.001). Apelin was negatively correlated to lactate dehydrogenase, indirect bilirubin, serum ferritin, end systolic diameter, tricuspid regurgitant jet velocity, right ventricle systolic pressure, pulmonary vascular resistance and tissue Doppler imaging S wave. Apelin cutoff value of 1650 ng/L could significantly detect the presence of cardiopulmonary complications in SCD with 90.9% sensitivity and 72.4% specificity.Apelin is a promising marker for screening of SCD patients at risk of cardiopulmonary disease because it is altered during the early subclinical stage of cardiac affection. A combination of apelin and echocardiography provides a reliable method to assess cardiopulmonary affection in young SCD patients.

Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use of LEN for augmentation of CAR-based immunotherapy in the clinical grounds.

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B‐cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally‐related pleomorphic highly‐aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine‐based second‐line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole‐exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53 , CREBBP , NUP214 , FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL‐restricted focal deletion of TP53 revealed that the pre‐MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre‐MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

Lecithin: retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs.